The goal of the Section on Human Genetics is to identify and study the function of mutated genes for human hereditary deafness. This work begins with the ascertainment of large families in which deafness appears to be inherited either as a dominant or a recessive trait. We then search for linkage of the deafness to 950,000 SNP markers across the human genome. During the past year we ascertained several large families segregating deafness, mapped a novel deafness locus, and identified a novel gene for nonsyndromic deafness DFNB86. Staff in the LMG have been working on the following projects, some of which were completed in the past year and have been published, are in press or are likely to be published in the near future. 1. Eleven years ago, DFNB32 was mapped to chromosome 1 by another research group, but the underlying gene was never reported. In four of our consanguineous families segregating recessively inherited nonsyndromic deafness linked to markers for the DFNB32 locus, we have identified three truncating mutations and a missense mutation in a gene in our refined DFNB32 interval. Ayesha Imtiaz, a graduate student, is exploring the function of the DFNB32 gene in the auditory system of a conditional ko mouse of this gene that we have engineered. As a collaboration with Dr. Katie Kindt, we are also constructing zebrafish models. 2. We will determine how noncoding mutations (del3 and del10 mutations) of HGF cause deafness DFNB39, which is the subject of ongoing work by Dr. Robert Morell. Hepatocyte growth factor is the 728 amino acid polypeptide product of the longest transcript arising from HGF (isoform 1). HGF activates downstream signaling only after proteolytic cleavage of the pro-HGFpolypeptide to form alpha and beta chains. The functions of HGF involve growth and wound repair, and somatic mutations of HGF have been implicated in some cancers. The 3'-UTR of isoform 6 transcripts include the deleted sequence of DFNB39 mutations. Despite a wealth of research on HGF, no comprehensive study of the temporal and spatial regulation of HGF isoforms has been made. Previously we described two different Hgf mouse models and demonstrated that both have hearing loss (Schultz et al., 2009). An Hgf transgene ubiquitously over-expresses full length HGF, and a conditional knockout of Hgf in which exon 5 was flanked by flox sequences. Taken together, these data indicate that dysregulation (too much or too little) of HGF causes hearing loss. To recapitulate deafness due to the del3 and del10 mutations in the 3-UTR of human HGF isoform 6, we have engineered an orthologous del10 deletion of the 3-UTR of Hgf isoform 6 in mouse. Using this mouse, we are exploring the functions of HGF in the auditory system. 3. We recently mapped a novel nonsyndromic deafness locus (DFNB81) to chromosome 19p, which is distinct from the closely linked DFNB72 locus (Rehman et al., 2011 EJHG). Mutations of GIPC3 are responsible for DFNB72 deafness. Using next-generation sequencing technology Atteeq rehman, PhD, a fellow in the LMG, identified mutations in CLPP encoding a mitochondrial chambered proteas. DFNB81 deafness was initially presumed to be nonsyndromic. However, further clinical chactareization of the affected subjects indicates Perrault syndrome characterized by hearing loss and female gonadal dysgenesis. This paper was published earlier in 2013 (Jenkinson, Rehman and Walsh et al., AJHG 2013). Atteeq is the co-first author and Friedman is the co-communicating author with William Newman, MD, PhD. Meghan Drummond, PhD, a fellow in the LMG/NIDCD, is continuing to work on the functions of CLPP in the auditory system. 4. In 2011 we mapped a novel locus for nonsyndromiic deafness to chromosome 16p. The locus was designated DFNB86 (Ali et al., 2011). Recently, Atteeq Rehman PhD, a fellow in the LMG, identified mutations of TBC1D24 as the cause of DFNB86 deafness. In collaboration with Suzanne Leal, PhD at Baylor College of Medicine and Andrew J. Griffith, MD, PhD, NIDCD/NIH we recently published this discovery (Rehman et al., 2014). 5. Grhl2-TMinsC/+ is a mouse model of DFNA28 human progressive hearing loss, which we reported ten years ago (Peters et al., 2002). A goal of our present study is to understand the function of the GRHL2 transcription factor in the auditory system. To that end, we have performed ChIP-Seq experiments using our own and commercially available antibodies to GRHL2, in a variety of tissues and cell types. In collaboration with Drs. Maria Ramirez and Saaket Varma at Boston University Medical School, and as a way to validate this technology in our laboratory, we extended our ChIP and expression analyses to include developing lung buds and the MLE15 cell line derived from adult mouse lung. GRHL2 is required for normal lung development. Varma et al. 2012, (Morell and Friedman are co-authors) showed that GRHL2 and NKX2-1 transactive each other and form a regulatory loop defining the critical transition from a type II cell type (cuboidal) to type I (squamous) in the lung alveolus. We are making a more comphrehensive survey of GRHL2 regulatory targets in the lung as compared to the auditory system using ChIP-Seq and our AB5500 sequencer. 6. Meghan Drummond PhD is working on the structure and dynamics of the gamma-actin and beta-actin in stereocilia using live-imaging and mouse mutants of gamma-actin, some of which are equivalent to alleles associated with dominantly inherited deafness DFNA20, a locus genetically mapped and reported by staff of the LMG several years ago and positionally cloned and identified as ACTG (under review). 7. 2014 NIDCD Core (DSC) is directed by Robert Morel, PhD. DSC personnel are Sahill Saxena, BS, and Keri Richard, BS. In FY2014, the NIDCD sequencing core (DSC) maintained and operated two sequencing machines: A 3730xl capillary sequencer and a HiSeq1000 sequencing machine. We also obtained a MiSeq sequencer. For the 3730xl sequencer, completed dye-terminator sequencing reactions are provided by individual users on 96-well plates. The plates are run for them on the 3730xl capillary sequencer and the raw data returned to the user. No data analysis service is provided. The DSC service is limited to the maintenance and operation of the capillary sequencer and occasional technical advice to users.Summary of 2014 AB3730xl capillary sequencer usage: 1,963 X 96 well plates sequenced For the Illumina HiSeq1000 sequencer, each user provides a DNA library diluted to 2 nM. The libraries are quantified on an Agilent BioAnalyzer 2100, then pooled to 10pM and clustered, along with PhiX controls, on an 8-lane flowcell. Sequencing is performed using SBS reagents in PE (paired end) format, usually 76 x 76, or 100 x 100, depending on application. The usage of the HiSeq100 was computed as the number of lane equivalents used by each laboratory. Summary of 2014 HiSeq1000 usage: 143 lanes were sequenced